Abnormal regulation of human deubiquitylating enzymes (DUBs) has been implicated in the pathogenesis of a number of human diseases including cancer, neurodegenerative disorders and viral infection. Ubiquitin-specific proteases (USPs) constitute the largest family among the five known DUB families. Human USPs are emerging as promising targets for pharmacological intervention. However, the lack of understanding of the specificity and regulation of USPs has hindered the progress in developing novel therapeutics. Remarkably, a recent global proteomic analysis of human DUBs revealed that over thirty human USPs are associated specifically with WD40-repeat proteins. Given its widespread occurrence, the interaction between WD40- repeat proteins and USPs likely represents a fundamentally important way of regulating USP activity. This application focuses on a prototypical USP WD40-repeat protein complex, human USP1/UAF1, and its close homologs. The overarching goal of this application is to gain an in-depth understanding of the specificity and regulation of human USPs and the USP WD40-repeat protein complexes. There are three specific aims: 1) Determine the energetics of the bipartite USP-Ub interaction in USP1 and USP2 (a structural homolog of USP1). Assess the contribution of the bipartite interaction to ubiquitin recognition and catalysis by double mutation analysis; 2) Probe the specificity of USP1/UAF1 using a physiological substrate and its close mimics. A new AlphaScreen-based assay will be developed to improve both the throughput and sensitivity of the deubiquitylation assay; 3) Determine the molecular mechanism by which UAF1 upregulates the activity of USP1 and the closely related human USP46. We will characterize the USP1/UAF1 and USP46/UAF1 complexes and map the interaction sites between UAF1 and the two USPs. The molecular basis for the stimulation of the USP enzymatic activity by UAF1 will be interrogated by enzymological approaches. Taken together, these studies have the potential to uncover important insights into the specificity and regulation of the prevalent USP WD40-repeat protein complexes. Further, our study will suggest new directions for pharmacologic intervention of this important class of human USPs.